Figure 1.
Postnatal inactivation of the dab1 gene leads to Tau hyperphosphorylation in hippocampal neurons.
A Phospho Tau levels at Ser202/Thr205 (AT8 site) and Ser 262 were increased in dab1cKI/cKI; ESR-CreERTM animals as compared to control dab1cKI/cKI animals that were treated with tamoxifen at P11 and sacrificed on P40. The presence of an APPSWE transgene did not increase the phospho Tau levels in dab1-deficient mice. Total Tau levels were relatively constant (5E2 immunoblot). Tau phosphorylation levels (AT8 site) normalized to total Tau were compared between hippocampal lysates of 3 sets of animals (* p = 0.01, Student's t-test). B Background levels of phospho Tau were observed by AT8 antibody immunohistochemical analysis of tamoxifen-treated (P7) dab1cKI/cKI hippocampi at P40. C Tau phosphorylation was observed in the soma of hippocampal neurons of dab1cKI/cKI; ESR-Cre mice treated in the same manner. D, E DAPI stained sections of tamoxifen-treated (P7) dab1cKI/cKI and dab1cKI/cKI; ESR-Cre hippocampi, respectively. Error bars indicate standard error of the mean (SEM) in all figures. Bar = 200 µm C, 100 µm E.
Table 1.
Strain-dependent expression of genes in the mouse hippocampus.
Table 2.
Characterization of genes that are differentially expressed between C57BL6 and BALB/c background Dab1 mutant mice and map to the Tau phosphorylation QTL on chromosome 1.
Figure 2.
Stk25 mRNA and protein levels are strain-dependent.
A Stk25 mRNA expression was 1.8-fold higher in C57BL/6 versus BALB/c strain in dab1 −/− and wild-type mice by real-time PCR. B Western blot analysis of Stk25 protein levels showed higher levels of expression in dab1 −/− and wild-type mutant animals on the C57BL/6 than the BALB/c strain. C Representative anti-Stk25 (upper panel) and anti-actin (lower panel) Western blots of hippocampal lysates.
Figure 3.
Stk25 knockdown reduces Tau phosphorylation in embryonic neurons.
A Phospho-Tau normalized to total Tau was significantly reduced in primary neurons infected with Stk25 shRNA viruses compared to neurons infected with either an empty vector (EV)-control or the control shRNA (con shRNA). B Tau phosphorylation at pSer202/pThr205 (AT8 antibody, 1st panel) was lower in samples with reduced Stk25 expression (3rd panel), whereas total Tau levels (2nd panel) and β-actin levels (4th panel) were unchanged. (* p<0.05, Student's t test, n = 3).
Figure 4.
Stk25 expression is not correlated with augmented Tau phosphorylation in a Tauopathy model or in transfected HeLa cells.
A Lysates from brains of 9.5 month old wild-type or Tg4510 mutant animals were immunostained for Stk25 and β-actin (upper panel). The average Stk25 signal normalized to β-actin from 3 separate samples from each genotype showed no statistically significant difference (lower panel). B Overexpression of Stk25-GFP did not lead to increased phosphorylation of co-transfected Tau in HeLa cells. Antibodies used for Westerns indicated at left: AT8 (phospho Ser202/Thr205 Tau) 5E2 total Tau. Sizes of expected Stk25-GFP and endogenous Stk25 are indicated at right.
Figure 5.
The Golgi extension and neuronal polarization phenotypes in dab1 mutant neurons is dependent upon mouse strain background.
The appearance of the Golgi apparatus was examined in 100 µm thick sections of the hippocampus at birth by immunostaining for GRASP65 and Ctip2 to identify pyramidal neurons. A In wild-type animals the Golgi apparatus extends several microns into the presumptive apical dendrite in the hippocampi, but in C57BL/6 dab1−/− mutants it is convoluted near the nucleus. The Golgi appears more elongated in the Balb/c dab1−/− mutant mice. Nucleus to Golgi distances were measured on isolated cells (insets). Arrowheads represent points used for measurements. B The nucleus to Golgi tip distances are greater for wild-type than dab1−/− mutants, and greater for BALB/c versus C57BL/6 dab1−/− mutants (*,** p<0.0001, Student's t test). C The number of multiple axon bearing neurons in dab1−/− mutant neurons is reduced on the BALB/c background as compared to the C57BL/6 background. In both cases, knocking down Stk25 leads to a further reduction in neurons with multiple axons and the development of neurons with no axons (* p<0.001 compared to the respective EV-control samples, ** p = 0.01 compared between EV-control samples, Student's t test) (Bar = 20 µm) Values for C57BL/6 samples have been published previously [34] and are shown here for comparison to BALB/c samples.
Table 3.
Sequence comparison of exon I of the APP gene from C57BL/6 and BALB/c mice.